The presence of positively charged groups, such as amine under acidic buffer conditions, quaternary amine functionality, or other electropositive groups in various analytes, is common. Mass spectrometry has been widely used to analyze such molecules in various modes, including MS2 or MS3 modes. Such applications are now standard practice for the analysis of clinically relevant analytes, such as peptides, proteins, and small molecules. Various mass tag reagents have been strategically designed that are or can be positively charged under acidic buffer conditions to conjugate or react with different classes of analytes and impart a positive charge to the overall molecule. Such tags can then be used to enhance signal intensity in positive ion mode analysis and for relative or absolute quantitation.
Unlike mass spectrometric analysis in positive mode, analysis in negative mode is not common. This disparity is partially due to a lack of electronegative groups or functionalities at our disposal. For example, carboxylic (—COOH), phosphoric (—OPO(OH)2), or sulfonic (—SO3H) functional groups can be ionized under basic conditions to produce negatively charged species, but the degree of ionization is highly dependent on the pH of the media. Mass spectrometric analysis under basic conditions has drawbacks, including limited variety and stability of volatile buffers and the instability of reversed phase HPLC columns under basic conditions.
There is an ongoing need for mass tag reagents that are suitable for use in negative ion mode mass spectrometry.